Mike+and+Aaron

Backround Info On Some Of The Equipment We Will Be using **High performance liquid chromatography** (or **high pressure liquid chromatography**, **HPLC**) is a form of colum Chromatography used frequently in Biochemistry and Analytical Chemistry to separate, identify, and quantify compounds based on their idiosyncratic polarities and interactions with the column's stationary phase. HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a charateristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. Editing By Aaron St.Onge

T he first step in the process we will most likely be doing ( Replace Soda with Coffee )

HPLC Part I  Modern HPLC instruments such as the Thermal Separation Products (TSP) HPLC you will be using to analyze caffeine and benzoate in soda pop look rather complicated. Most are computer controlled, have multi-channel pumps, integral solvent degassers and gradient mixers, auto samplers and two or more types of detector. Although convenient for the user, these 'extras' are not really necessary for producing high quality separations. A pump, detector and a visual output are all that is really needed. Your assignment for part I is to assemble our 'bare bones' HPLC and produce a chromatogram showing the separation of a standard mixture of caffeine and benzoate.

Notes  Use a mobile phase of 50:50 methanol:water buffered to pH 3 with 10 mM phosphate. Filter and degas the mobile phase. Prepare a solution of 0.10 mg/ml caffeine and 0.30 mg/ml benzoate in diH2O. Be sure to purge all lines with mobile phase before attaching the column. Use a detection wavelength of 254 nm. Allow the column to equilibrate for at least 30 minutes at 1ml/min before performing the separation. After the separation wash the column with 100 % water for 30 minutes at 1 ml/min.